mmg_233_2013_genetics_genomicswikiaorg-20200214-history
Viruses of Amoebae may be Human Pathogens: Metagenomic Evidence
Since their original discovery in 2003, Mimiviruses and numerous other amoebal pathogens (including Megaviridae and Marseilleviridae) have been retrospectively identified in human microbiota metagenomic studies. Though there is a clear dearth of research concerning the life cycles and pathogenicity of many of these recently discovered giant viruses, clinical evidence suggests that Mimivirus is a human pathogen capable of causing pneumonia. This claim is substantiated by metagenomic findings (1, 2). Mimiviruses and other Amoebal Viral Pathogens Though mimiviruses were not classified until 2003, they were actually isolated 20 years previously in England during an enigmatic 1992 pneumonia outbreak in Bradford (1, 3). Researchers at the Public Health Laboratory in Leeds had previously used Acanthamoeba species to isolate several bacterial etiological agents of pneumonia (i.e. Legionella pneumophila) from human samples. These phagocytic protists are ubiquitous in the environment and have "been described as true Trojan horses because they can host multiple human pathogenic bacterial that survive and multiply within the amoeba and are protected from various external physical and chemical agents when the amoeba encyts (1)". Therefore, when trying to isolate the (what they assumed to be) bacterial etiological agent behind the pneumonia outbreak in Bradford, researchers collected water from nearby water cooling towers as they believed these to be harboring the culpable pneumonia pathogens and inoculated amoeba. When monitoring the cultured amoeba, the researchers took the formation of lysis plaques to indicate the presence of an amoebal pathogen. Several new Legionella species were isolated and characterized during the investigation as was a previously unobserved strain of what appeared to be a Gram-positive coccus, which was aptly named the Bradford coccus (1). But when researchers later tried to investigate the physiological and anatomical characteristics of this new Bradford coccus, they met several insurmountable barriers; the new pathogen's genome did not seem to be susceptible to PCR amplification and the researchers were unable to sequence the coccus's 16S ribosomal DNA. These unanticipated failures lead the researchers to try a new approach: electron microscopy. When viewed with the electron microscope, the Bradford coccus was revealed to the icosahedral structure classical of many known viruses. Subsequent studies including genomic sequencing and analysis proved the viral nature of the "Bradford coccus", which was then renamed "Mimivirus", a name derived from "mimicking microbe" (1, 3). Since Mimiviruses were characterized in 2003, numerous other intra-amoebal NCLDVs have been described (2). Viral Structure Mimiviruses, Marseilleviruses, Megaviruses and other giant amoebal viruses all possess an icosahedral capsid with multiple projecting proteinous filaments. Thus far, all characterisized intra-amoebal parasites have been placed within the nucleocytoplasmic large DNA virus (NCLDV) group, which also contains the viral family poxviridae and thus the etiological agent behind small pox. All viral families that fall within this group possess similar virion structures and enormous, double-stranded DNA genomes. Individually, mimiviruses's genome is composed of over a million base pairs and is speculated to encode about 911 protein-coding genes, which is quite a few when one considers the fact that influenza's genome only contains about 11 open reading frames or protein-coding genes. Interestingly enough, the mimivirus genome is also known to contain a number of open reading frames that are bizarrely homologous to previously characterized genes known to encode translational proteins (i.e. aminoacyl-tRNA synthethases and translation initiation factors) despite its viral nature. This and other similar findings in other viral families have led researchers to question how viruses are currently classified and thought of in contemporary scientific literature. Pathogenicity Though there is little to no evidence currently in existence to identify Marseilleviruses as pathogenic to humans, one can decisively classify Mimivirus as a pneumonic pathogen as it has met all 4 of Koch's Postulates. Dr. Philippe Colson reports that "Mimivirus has never been isolated outside a pathological context in human respiratory samples, it was isolated from pneumonia patients without finding any other causative agent of this pathology (1)". Over the last decade, 2 experimental studies exploring the pathogenicity of Mimivirus have been preformed. The results of one such study suggests that Mimiviruses infect and replicated within macrophages. The other used a murine model to evaluate the pneumonic potential of this colossal amoebic virus, finding that mice infected intracardically with Mimivirus developed pneumonia. The subsequent isolation of Mimivirus from the lungs of the infected mice conclusively proved that the pneumonia was of Mimivirus etiology (1). Various studies preformed over the last decade have used serological assays paired with microimmuno-fluorescence techniques to determine the prevalence of Mimivirus infections in pneumonia patient populations. In many of these studies, the IgG prevalence observed in pneumonia patients typically fell within a 10 to 20% range, though no Mimivirus IgG were detected in those suffering from aspiration pneumonias. A study published by Bousbia et al in 2013 provided compelling evidence that implicates Mimiviruses as one of the etiological agents behind nosocomial pneumonic infections (1). Mimivirus DNA has only been detected twice in the bronchoalveolar fluid of a pneumonia patient using PCR analysis, but this is likely due to the high degree of heterogeneity between different Mimivirus species (1, 2). Mimiviruses have been identified in pathological processes other than pneumonia as well. In 2010, Lentillevirus, a new strain of Mimivirus, was isolated in the contact lens storage case liquid of a "17-year-old myopic woman presenting with keratitis (1)". When no bacterial or Acanthamoeba organisms were detected in the young woman's corneal scrapping, the researchers involved turned to her contacts case, where upon they isolated several bacterial pathogens, Acanthamoeba polyphaga and two previously unknown viral agents. One of these viral pathogen was later identified to be a member of the Mimivirus viral family and named Lentillevirus while the other was subsequently identified as a new virophage and named Sputnik 2. A serological assay conducted on a couple experiencing asthenia, gastrointestinal distress and myalgia and a low grade fever detected the presence of antibodies specific to the Sputnik virophage of Mimivirus, a finding that was later confirmed using two-dimensional gel electrophoresis and matrix assisted laser desorption ionization mass spectrometry. Because serum samples had been collected from the woman 5 months earlier due to her pregnancy, it could be definitively stated that she at least exhibited seroconversion for the Sputnik virophage (1). Metagenomic evidence Researchers in France (the very same researchers who published the 2 articles cited in this treatise) preformed a metagenomic study on the stools of an apparently healthy 20-year old man living in rural Senegal. The researchers employed 2 separate ultra-deep sequencing PCR techniques in an attempt to isolate all bacterial 16S ribosomal DNA (rDNA) present. In addition to the classical PCR method, a new method based on the PCR amplification of the V6 region of the 16S rDNA through the use of the universal primers 917F and 1391R to avoid PCR amplification bias. This new technique first required the complete enzymatic digestion of the DNA within the fecal specimen using the restriction endonucleases EcoO1901 and BrsG1 as these endonucleases are known to cleave sites within the V6 16S rDNA region, generating fragments known to contain the VS 16S rDNA regions. This would ensure that the DNA subjected to subsequent PRC amplification contained sequences complementary to the primers used. The products generated using the new and classical PCR methods were then sequenced using the 454 FLX Titanium instrument, an automated system that employs 454 pyrosequencing, and the sequences generated were analyzed using BLAST and the QIIME pipeline. Among the reads generated by the enzymatic digestion, less than one percent were related to known bacterial 16S rDNA sequences. The researchers then analyzed the remaining 99.9% of the reads generated for sequences related to known Mimivirus and Marseillevirus genomes using CLC bio software. Default CLC analysis parameters (50% minimum coverage, 80% minimum similarity) were employed in this inquiry. The French researchers also preformed tBLASTn with the giant viruses against the read sequences (2). The metagenomic reads recovered from the fecal specimen collected from a young Senegalese were found to contain 44 and 9 sequences that could be mapped to the Mimivirus and Marseillevirus genomes, respectively. Amazingly, 12 reads more than 300 base pairs in length could be mapped to the Mimivirus genome with more than 90% identity and coverage and tBLASTn searches with the Mimivirus and Marseillevirus genomes yielded more 2,306 and 259 hits, accordingly. These extraordinary findings prompted the researchers to inoculate an Acanthamoeba polyphaga culture with 1 gram of the fecal specimen collected so that the presence of the amoebal viruses could be confirmed. From this amoebal culture, a formerly unknown Marseillevirus, dubbed Senegalvirus, was isolated and subjected to various genomic and proteomic analyses. Comparative genomic studies coupled with family-B DNA polymerase analyses allowed the researchers to construct a phylogeny that placed Senegalvirus with Marseillevirus within the Marseilleviridae viral family. The results of this study suggest that Marseilleviruses and other Megaviruses (i.e. Mimivirus) could be more prevalent in the human microbiome than previously imagined (1, 2). In a later study, the same French researchers collected viral metagenomic reads from eleven published studies and obtained additional metagenomic data from human samples (including stool, nasopharyngeal aspirates, saliva, oropharyngeal swabs and sputum) with the intention of running all reads generated against the NCBI Gen-Bank protein sequence database to assess the prevalence of Megaviruses (e value threshold used: 1e-5). In this particular analysis, only hits with alignment lengths greater than 40 amino acids were considered. Metagenomic reads from eleven additional studies were also collected and "annotated with an in-house strategy using BLASTn searches against all genomes of the Megavirales members and virophages available in the NCBI GenBank sequence database as well as those not yet released. available the researcher's laboratory (2)". The sequences used in this later analysis were first processed using Prinseq and other techniques so that all duplicated, low quality and low complexity reads could be excluded from subsequent analyses. Several metagenomic reads from stool and oropharyngeal virome samples were found to match Mimivirus sequences, though none of the ensuing BLAST searches preformed on these sequences identified Mimivirus as a top hit. Mimivirus proteins were typically among the best BLAST hits however, with e-values ranging from 1e-6 to 1e-14 and amino acid identities ranging from 28 to 58% (2). Of particular interest was a 130 nucleotide read recovered from a pulmonary microbiota metagenomic data set collected from patients suffering from acute exacerbation of idiopathic pulmonary fibrosis. When this sequence was run through BLASTn, BLASTx and tBLSTx searches, an enzyme unique to the Mimivirus COV was found to be the best match in all 3 instances. Another crucial finding reported by the researchers was generated when 4 human gut metagenomes were subjected to BLASTn analysis. This search produced 384 viral genomic hits, 299 of which were Mimivirus. Though further research is certainly required, this and other research not discussed suggests that Megaviruses are ubiquitous in the environment and that these viruses are capable of inducing disease in humans (1, 2). References 1. Giant Viruses of Amoebae as Potential Human Pathogens 2. Evidence of the megavirome in humans 3. Mimivirus